Monolithic capillary columns based on pentaerythritol acrylates for molecular‐size‐based separations of synthetic polymers

Monolithic capillary columns based on pentaerythritol triacrylate and pentaerythritol tetraacrylate were synthesized using different compositions of polymerization mixtures and different polymerization conditions. The impact of porogen type and porogen/monomer ratio on the porosity of synthesized monoliths was investigated. Porogen type appears to be the main factor influencing the separating properties of the monolithic sorbent. Using optimal polymerization conditions (porogen type, porogen/monomer ratio, reaction temperature, time etc.) monoliths with a porous structure optimized for polymer separations can be obtained. The monolithic capillary columns containing porous sorbents with optimized porosity are capable of separating 10 to 12 polystyrene standards in one chromatographic run utilizing both size exclusion chromatography and hydrodynamic chromatography separation mechanisms.     

Columns switch recycling size exclusion chromatography for high resolution protein separation

Columns switch recycling size exclusion chromatography (csrSEC) was proposed to achieve high resolution protein separation with good biocompatibility. Proteins were firstly separated by two serially coupled SEC columns, and fractions were in sequence switched back to the first column by two-position valves for further separation in terms of close-loop recycling until satisfactory resolution was achieved. Compared to SEC, the separation window was broadened by increasing column length via cycling without further increase on back pressure. Compared to recycling SEC (rSEC), the overtaking of later eluted components by early eluted ones after several cycles could be avoided for complex sample analysis, by parking fractions in the second SEC column before transferred in turn back to the first one for cycling ordinally. In our experiments, the baseline separation of five proteins with molecular weight ranging from 10 kDa to 80 kDa was achieved by csrSEC. Furthermore, a multidimensional csrSEC–RPLC platform was constructed, and peak capacity up to 3600 was obtained for protein separation. All these results demonstrated that csrSEC is a promising protein separation mode with good biocompatibility, broadened separation window and improved resolution.     

体积排阻色谱法测定胸腺肽分子量校准曲线条件的优化

在体积排阻色谱(SEC)法测定胸腺肽分子量校准曲线过程中,流动相中乙腈的比例对核糖核酸酶A、人胰岛素、胸腺肽α1和生长激素释放抑制因子4种蛋白的保留时间有重要影响,进而影响校准曲线的线性关系。当乙腈比例为75%时,胸腺肽分子量校准曲线线性最好,此时分子量校准方程为y=-3.138 6x+21.724,线性相关系数r2=0.988 5。4种蛋白的理论塔板数在45 783～63 345之间,拖尾因子在0.96～1.18之间,分离度在3.52～8.82之间。SEC法测定胸腺肽分子量校准曲线的液相色谱条件对4种蛋白的分离效果优异,分子量校准曲线线性良好,可用于胸腺肽制剂中高分子量物质的检测。     

Detailed characterization of cationic hydroxyethylcellulose derivatives using aqueous size-exclusion chromatography with on-line triple detection

A more complete understanding of polymeric, cationic cellulose derivatives, including polyquaterium-10 (Polymer JR), has become increasingly important in the eye care industry as thorough characterization of raw materials helps promote product quality and process control. Often such detailed information requires utilization of a combination of analytical techniques. In this work three Polymer JR samples with different viscosities were characterized using aqueous size exclusion chromatography (SEC) with a light scattering detector, a differential viscometer, and a differential refractometer (triple detection). Detailed molecular information such as absolute molecular weights, molecular weight distributions, intrinsic viscosities, and molecular conformations were obtained. One major challenge of analyzing cationic polymers is abnormal size exclusion separation, which could be caused by the ionic interaction between sample molecules and the column packing material. A selection of mobile phases varying in pH, buffer, organic solvent content, and molar concentration of salts was employed to evaluate the correlation of obtained molecular weight values and mobile phase composition. Universal calibration concept was used to examine the abnormal size exclusion separation phenomenon of Polymer JR samples when using different mobile phases. It was observed that the abnormal size exclusion was dependent on both the separation conditions and molecular weights of the samples. Despite the changes in separation parameters and uncharacteristic polymeric structure compared to conventional SEC samples, the use of aqueous SEC with triple detection provided reproducible and valuable molecular information of Polymer JR samples with low to medium molecular weights. By using a combination of high buffer content and adding organic solvent, the abnormal exclusion separation of high molecular weigh Polymer JR could be considerably reduced.     

Characterization of poly(ethylene glycol)-b-poly(ε-caprolactone) by liquid chromatography under critical conditions: Influence of catalysts and reaction conditions on product composition

Amphiphilic block copolymers were synthesised by ring opening polymerization of ε-caprolactone with polyethylene glycol monomethyl ether (PEG-MME) using different catalysts (boron trifluoride, sodium hydride, and tin octoate) in a one pot procedure. The products obtained were characterized with respect to their molar mass distribution and content of homopolymers using size exclusion chromatography (SEC), Liquid chromatography under critical conditions (LCCC) and MALDI-TOF-MS. The homopolymers of caprolactone could be separated from the block copolymer by LCCC on a reversed phase column in tetrahydrofurane-water mobile phases with evaporative light scattering detection (ELSD). Residual monomer could be determined under the same conditions using density detection, a separation of the copolymer from residual initiator could be achieved on a normal phase column in acetone-water mobile phases.     

Evaluation of silica gel-immobilized phosphorylcholine columns for size exclusion chromatography and their application in the analysis of the subunit structures of fish-egg lectins

Columns of phosphorylcholine (PC) immobilized on silica gel were shown to be useful for size exclusion chromatography (SEC) of proteins. The columns provided good separation of proteins in 50 mM sodium phosphate buffer (pH 6.9) containing 0.25 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights with a correlation coefficient (R²) of 0.978–0.992. The columns were used in analyzing the subunit structures of the rhamnose-binding lectins CSL1, CSL2, and CSL3, isolated from chum salmon (Oncorhynchus keta) eggs. Although the lectins, which are a group of carbohydrate-binding and hydrophobic proteins, behaved anomalously in SEC with conventional matrices, they could be eluted from the immobilized PC columns without non-size-related retention, thereby allowing their molecular weights to be reliably estimated.     

原子转移自由基聚合制备聚(丙二醇-g-苯乙烯)

以氯甲基化苯氧基聚丙二醇 (CMPOPPG)为大分子引发剂 ,由CuCl/bpy催化的苯乙烯原子转移自由基聚合反应合成了聚 (丙二醇 g 苯乙烯 ) .CMPOPPG经环氧丙烷 (PO)与缩水甘油苯基醚 (GPE)的开环聚合和氯甲基化反应制得 .接枝聚合反应具有可控性 .用1H NMR和微库仑分析法对接枝共聚物进行了表征 .结果表明 ,支链分子量可控 ,接枝率可达 8.6.     

Characterization of EO-PO Block Copolymers by Liquid Chromatography Under Critical Conditions

Summary: Block copolymers of ethylene oxide (EO) and propylene oxide (PO) are characterized by liquid chromatography under critical conditions (LCCC) for EO. At the critical adsorption point (CAP) for one structural unit, the non-critical block can elute in size exclusion (SEC) or adsorption (LAC) mode. Depending on the molar mass and architecture of the polymers, different strategies are applied. For samples with a higher molar mass, the SEC separation is the method of choice, while lower molar masses also allow a LAC separation. Examples for both situations are given, which show, that these approaches yield different information. In the SEC mode, homopolymers and diblocks can be separated from the triblocks. In LAC mode, a baseline resolution of individual oligomers can be achieved, in which homopolymers, diblocks and triblocks with the same number of repeat units of the non-critical block have the same elution volume.     

Amphiphilic polymers based on higher alkylene oxides: Synthesis and characterization by different chromatographic techniques

Homopolymers and block copolymers of higher epoxides (butene oxide and hexene oxide) are synthesized using 1-alkanols and polyethylene glycol monomethyl ether (PEG-MME) 1100 as initiators by anionic ring opening polymerization in bulk. Most of the samples were synthesized with controlled microwave heating in sealed vessels. Tri- and tetrablock copolymers with different repeat units in the individual blocks are synthesized by living polymerization with addition of the next monomer after complete consumption of the previous one. The products thus obtained are characterized using size exclusion chromatography (SEC), liquid chromatography under critical conditions (LCCC) and liquid adsorption chromatography (LAC).     

Polymeric cation-exchange monolithic columns containing phosphoric acid functional groups for capillary liquid chromatography of peptides and proteins

Two different monoliths, both containing phosphoric acid functional groups and polyethylene glycol (PEG) functionalities were synthesized for cation-exchange chromatography of peptides and proteins. Phosphoric acid 2-hydroxyethyl methacrylate (PAHEMA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) were reacted with polyethylene glycol diacrylate (PEGDA) and polyethylene glycol acrylate (PEGA), respectively, in 75-μm i.d. UV-transparent fused-silica capillaries by photo-initiated polymerization. The hydrophobicities of the monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. The resulting monoliths exhibited lower hydrophobicities than strong cation-exchange monoliths previously reported using PEGDA as cross-linker. Dynamic binding capacities of 31.2 and 269 mg/mL were measured for the PAHEMA–PEGDA and BMEP–PEGA monoliths, respectively. Synthetic peptides were eluted from both monoliths in 15 min without addition of acetonitrile to the mobile phase. Peak capacities of 50 and 31 were measured for peptides and proteins, respectively, using a PAHEMA–PEGDA monolith. The BMEP–PEGA monolith showed negligible hydrophobicity. A peak capacity of 31 was measured for the BMEP–PEGA monolith when a 20-min salt gradient rate was used to separate proteins. The effects of functional group concentration, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run [relative standard deviation (RSD) < 1.99%] and column-to-column (RSD < 5.64) reproducibilities were achieved. The performance of the monoliths in ion-exchange separation of peptides and proteins was superior to other polymeric monolithic columns reported previously when organic solvents were not added to the mobile phase.     

多维高效液相色谱分离模式组合

简述了多维高效液相色谱法的特点及发展简况,重点对分子排阻色谱／反相色谱、离子交换色谱／反相色谱、正相色谱／反相色谱、分子排阻色谱／离子交换色谱、液固色谱／反相色谱、亲合色谱／反相色谱、非手性柱／手性柱等的联用模式及实际应用进行了概括和总结。     

Monoliths from poly(ethylene glycol) diacrylate and dimethacrylate for capillary hydrophobic interaction chromatography of proteins

Rigid monoliths were synthesized solely from poly(ethylene glycol) diacrylates (PEGDA) or poly(ethylene glycol) dimethacrylates (PEGDMA) containing different ethylene glycol chain lengths by one-step UV-initiated polymerization. Methanol/ethyl ether and cyclohexanol/decanol were used as bi-porogen mixtures for the PEGDA and PEGDMA monoliths, respectively. Effects of PEG chain length, bi-porogen ratio and reaction temperature on monolith morphology and back pressure were investigated. For tri- and tetra-ethylene glycol diacrylates (i.e., PEGDA 258 and PEGDA 302), most combinations of methanol and ethyl ether were effective in forming monoliths, while for diacrylates containing longer chain lengths (i.e., PEGDA 575 and PEGDA 700), polymerization became more sensitive to the bi-porogen ratio. A similar tendency was also observed for PEGDMA monomers. Polymerization of monoliths was conducted at approximately 0 °C and room temperature, which produced significant differences in monolith morphology and permeability. Monoliths prepared from PEGDA 258 were found to provide the best chromatographic performance with respect to peak capacity and resolution in hydrophobic interaction chromatography (HIC). Detailed study of these monoliths demonstrated that chromatographic performance was not affected by changing the ratios of the two porogens, but resulted in almost identical retention times and comparable peak capacities. An optimized PEGDA 258 monolithic column was able to separate proteins using a 20-min elution gradient with a peak capacity of 62. Mass recoveries for test proteins were found to be greater than 90, indicating its excellent biocompatibility. All monoliths demonstrated nearly no swelling or shrinking in different polarity solvents, and most of them could be stored dry, indicating excellent stability due to their highly crosslinked networks. The preparation of these in situ polymerized single-monomer monolithic columns was highly reproducible. The relative standard deviation (RSD) values based on retention times of retained proteins were all within 2.2%, and in most cases, less than 1.2%. The RSD values based on peak areas were within 9.5%, and in most cases, less than 7.0%. The single-monomer synthesis approach clearly improves column-to-column reproducibility.     

Simple capillary flow porometer for characterization of capillary columns containing packed and monolithic beds

A simple capillary flow porometer (CFP) was assembled for through-pore structure characterization of monolithic capillary liquid chromatography columns in their original chromatographic forms. Determination of differential pressures and flow rates through dry and wet short capillary segments provided necessary information to determine the mean diameters and size distributions of the through-pores. The mean through-pore diameters of three capillary columns packed with 3, 5, and 7 μm spherical silica particles were determined to be 0.5, 1.0 and 1.4 μm, with distributions ranging from 0.1 to 0.7, 0.3 to 1.1 and 0.4 to 2.6 μm, respectively. Similarly, the mean through-pore diameters and size distributions of silica monoliths fabricated via phase separation by polymerization of tetramethoxysilane (TMOS) in the presence of poly(ethylene glycol) (PEG) verified that a greater number of through-pores with small diameters were prepared in columns with higher PEG content in the prepolymer mixture. The CFP system was also used to study the effects of column inner diameter and length on through-pore properties of polymeric monolithic columns. Typical monoliths based on butyl methacrylate (BMA) and poly(ethylene glycol) diacrylate (PEGDA) in capillary columns with different inner diameters (i.e., 50–250 μm) and lengths (i.e., 1.5–3.0 cm) were characterized. The results indicate that varying the inner diameter and/or the length of the column had little effect on the through-pore properties. Therefore, the through-pores are highly interconnected and their determination by CFP is independent of capillary length.     

Polymer characterization by interaction chromatography

Liquid chromatography (LC) is a powerful tool for the characterization of synthetic polymers, that are inherently heterogeneous in molecular weight, chain architecture, chemical composition, and microstructure. Of different versions of the LC methods, size exclusion chromatography (SEC) is most commonly used for the molecular weight distribution analysis. SEC separates the polymer molecules according to the size of a polymer chain, a well‐defined function of molecular weight for linear homopolymers. The same, however, cannot be said of nonlinear polymers or copolymers. Hence, SEC is ill suited for and inefficient in separating the molecules in terms of chemical heterogeneity, such as differences in chemical composition of copolymers, tacticity, and functionality. For these purposes, another chromatographic method called interaction chromatography (IC) is found as a better tool because its separation mechanism is sensitive to the chemical nature of the molecules. The IC separation utilizes the enthalpic interactions to vary adsorption or partition of solute molecules to the stationary phase. Thus, it is used to separate polymers in terms of their chemical composition distribution or functionality. Further, the IC method has been shown to give rise to much higher resolution over SEC in separating polymers by molecular weight. We present here our recent progress in polymer characterization with this method. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 1591‐1607, 2005     

以氯甲基聚苯乙烯为基质的新型排阻色谱固定相的合成及在生物大分子分离中的应用

以单分散交联聚氯甲基苯乙烯为基质,先后与乙二胺和δ－葡萄糖酸内酯反应,得到了亲水性能良好的排阻色谱固定相,其表面羟基含量高达５．４ｍｍｏｌ／ｇ。本文评价了改性后树脂的亲水性、溶剂匹配性、耐压性能及化学稳定性,用三羟甲基氨基甲烷（Ｔｒｉｓ）缓冲溶液（ｐＨ＝７．０）为流动相,蛋白质,混合样的分离遵循排阻色谱分离机理。聚苯乙烯和蛋白质标准样的排阻色谱分离机理。聚苯乙烯和蛋白质标准样的排阻色谱校正曲线 范     

Separation of statistical poly[(N-vinyl pyrrolidone)-co-(vinyl acetate)]s by reversed-phase gradient liquid chromatography

Although size exclusion chromatography (SEC) has been used successfully to determine the molecular weight distribution (MWD) of statistical poly[(N-vinyl pyrrolidone)-co-(vinyl acetate)]s [PVPVAs], SEC cannot separate the copolymers according to their chemical composition. In this article, the separation of commercial PVPVAs with varying chemical compositions is reported, by aqueous reversed-phase gradient liquid chromatography (RPLC) using polystyrene-divinylbenzene-based wide pore columns. RPLC-SEC cross-fractionation indicates the presence of molar mass dependant effects during RPLC separation due to broad MWD for the copolymer studied; therefore the width of the RPLC peak could not be associated entirely with chemical composition distribution of the copolymer. Coupling of RPLC with online FTIR spectroscopy reveals the increase of VA content with increasing THF gradient, an indication of interaction mechanism between VA repeating units and the stationary phase for water soluble PVPVAs. Separation of water insoluble PVPVAs and PVAs by the RPLC are possibly based on both interaction and precipitation/redissolution mechanisms.     

Analysis of complex phthalic acid based polyesters by the combination of size exclusion chromatography and matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used in conjunction with size exclusion chromatography (SEC) to investigate a model polyester system based on phthalic anhydride–1,2-propylene glycol. The polyesters were synthesized with a 30% molar excess of glycol, with kinetic samples being removed during different intervals of the polyesterification reaction. SEC was used to track the course of the reaction by determining the molecular weight and molecular weight distributions before subsequent off-line coupling with MALDI-TOF MS as a selective detection method to determine the chemical composition, identify the functionality type distributions as well as assist in assigning structural conformations. Mass spectrometry analysis proved to be a highly effective tool to facilitate the identification of the narrowly dispersed fractions obtained from the chromatographic separations as well as serve as a core method to investigate the heterogeneous nature of the bulk kinetic samples. Through the hyphenation of these sophisticated polymer characterization techniques, information on the molecular heterogeneity of the model polyesters, showing a complex variety of possible distributions, was obtained.     

Characterization of polyethers using different methods: SFC,LAC and SEC with different detectors,and MALDI-TOF-MS

Polyethylene glycol 300 (PEG 300) and polypropylene glycol 425 (PPG 425) were analyzed using size exclusion chromatography (SEC) in different solvents, normal and reversed phase liquid chromatography (NPLC and RPLC), supercritical fluid chromatography (SFC), and matrix-assisted laserdesorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). In liquid chromatography, different detectors were used: density, refractive index (RI) and evaporative light scattering detector (ELSD).     

Preparation and characterization of lauryl methacrylate-based monolithic microbore column for reversed-phase liquid chromatography

Poly(lauryl methacrylate-co-ethylene dimethacrylate) monoliths were in situ synthesized within the confines of a silicosteel tubing of 1.02 mm i.d. and 1/16" o.d. for microbore reversed-phase HPLC. In order to obtain practically useful monoliths with adequate column efficiency, low flow resistance, and good mechanical strength, some parameters such as total monomer concentration (%T), cross-linking degree (%C) and polymerization temperature were optimized. High-efficiency monoliths were successfully obtained by thermal polymerization of a monomer mixture (40%T, 10%C) with a binary porogenic solvent consisting of 1-propanol and 1,4-butandiol (7:4, v/v) at a high temperature of 90 °C. The morphology and porous structure of the resulting monoliths were assessed by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC), while the column performance was evaluated through the separations of a series of alkylbenzenes in acetonitrile-water (50:50, v/v) eluent. At a normal flow rate of 50 μL/min (corresponding to 1.66 mm/s), the optimized monolithic columns typically exhibited theoretical plate numbers of 6000 plates/10 cm-long column for amylbenzene (k>40), and the pressure drop was always less than 1 MPa/10 cm. The monoliths, which were chemically anchored to the tube inner wall surface using a bifunctional silylation agent, exhibited adequate mechanical strength of up to 12-13 MPa, and were properly operated at 10 times higher flow rate than normal, reducing the separation time to one tenth. The lauryl methacrylate-based monolithic column was applied to a rapid and efficient separation of ten common proteins such as aprotinin, ribonuclease A, insulin, cytochrome c, trypsin, transferrin, conalbumin, myoglobin, β-amylase, and ovalbumin in the precipitation-redissolution mode. Using a linear CH(3)CN gradient elution at a flow rate of 500 μL/min (10-times higher flow rate), 10 proteins were baseline separated within 2 min.     

Preparation of polymeric monoliths by copolymerization of acrylate monomers with amine functionalities for anion-exchange capillary liquid chromatography of proteins

Two novel polymeric monoliths for anion-exchange capillary liquid chromatography of proteins were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion-exchanger and quaternary amine as a strong anion-exchanger, respectively. An alternative weak anion-exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. Important physical and chromatographic properties of the synthesized monoliths were characterized. The dynamic binding capacities of the three monoliths (24 mg/mL, 56 mg/mL and 32 mg/mL of column volume, respectively) were comparable or superior to values that have been reported for various other monoliths. Chromatographic performance was also similar to that provided by a modified poly(GMA-ethylene glycol dimethacrylate) monolith. Separation of standard proteins was achieved under gradient elution conditions using these monolithic columns. Peak capacities of 34, 58 and 36 proteins were obtained with analysis times of 20–30 min. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, additional surface modifications were avoided and the PEGDA crosslinker helped to improve the biocompatibility of the monolithic backbone.